Objectives Recently, it was discovered that Methylobacterium extorquens possesses a spectrum of phenotypic states conferring non-genetic resistance to formaldehyde. The non-genetic resistance allowed rapid growth on levels of formaldehyde which were lethal to the majority of the population. To investigate whether this is a common phenomenon, we tested the distantly related Escherichia coli for heterogeneity to formaldehyde stress during growth on glucose. Results Like M. extorquens, E. coli populations have a wide, reversible, continuous range of non-genetic resistance to formaldehyde. However, there were differences from what was found for M. extorquens. Most E. coli growth occurred after formaldehyde levels had dropped, suggesting that an effect similar to antibiotic persistence could be the cause. We found the dormant cell state can maintain formaldehyde resistance through the lack of dilution of the proteome caused by growth. These data suggest that dormancy can preserve phenotypic heterogeneities in other traits.

Heritable non-genetic phenotypic variation-broadly, epigenetics-can potentially influence evolutionary outcomes as direct targets of selection or through interactions with genetic variation. While their evolutionary benefits in generating phenotypic diversity in changing environments are well-characterized, there has been relatively little consideration of how the joint influence of epigenetic changes and mutations would affect traversal of multi-peak adaptive landscapes. Here, we discover general principles for how epigenetics, by generating an epigenetic quasispecies (clusters of semi-stable phenotypes mapped to a single genotype), tends to improve adaptive outcomes of an asexual population on rugged fitness landscapes even in a constant environment. In particular, rapid epigenetic changes tend to smooth out suboptimal fitness peaks through incorporating fitness contributions of epimutations, allowing access to better adaptive outcomes. Remarkably, the average impact of epigenetics is more strongly influenced by an approximate balance between switching rates rather than the absolute rate at which switching occurs. These findings demonstrate that epigenetic changes can be influential even without having strong heritability and have a striking, yet generally invisible, beneficial role in shaping a population's adaptive trajectory.

Fitness tradeoffs between different environments enable the maintenance of microbial diversity. While the importance of tradeoffs is clear, it has been surprisingly difficult to predict which traits they will occur between and at how granular a level. For example, it is unclear whether performance between a constant versus pulsed exposure of the same stress tends to be positively correlated, independent of each other, or negatively correlated. Empirically, it has been shown that a critical feature structuring microbial communities is temperature. However, the compatibility between strategies to deal with different forms of heat stress is unclear. For instance, are strains that grow well at higher temperatures also stronger at withstanding heat shock? To understand how environmental microbes can adapt to better deal with heat stress, we performed an evolution experiment using a dominant phyllosphere microbe Methylobacterium extorquens in a regime of intermittent heat shock. We identified the genetic basis of adaptation, discovering a large number of loci capable of mediating adaptation to heat shock, many of which had not been previously linked to heat stress. Despite the genetic divergence among evolved isolates, we discovered a general tradeoff between heat shock resistance and growth at consistently elevated temperatures. We found this tradeoff was not limited to evolved isolates, but also represented across a sample of environmentally isolated Methylobacterium strains. These findings indicate a generic conflict between strategies to deal with heat shock recovery and growth at elevated temperatures, suggesting even variation in intensities of a stressor can drive diversity in microbial strategies.IMPORTANCEOne of the key forces shaping the microbial diversity in nature is temperature. However, temperature in ecological settings is variable, and it is unknown if strategies to deal with different intensities of high temperature are compatible or not. Using evolution experiments, we identify the genetic basis of adaptation to heat shock in Methylobacterium extorquens, a dominant member of the phyllosphere microbiome. We discover a number of genetic targets where beneficial mutations improve heat shock resistance, most of which have not been implicated with heat stress before. For both the evolved isolates and a set of environmentally isolated Methylobacterium strains, we discover a general tradeoff between recovery after heat shock and growth at elevated temperatures. While the strategies to deal with increasing temperatures have garnered significant interest, our results suggest that even different intensities of heat stress can select for distinct and incompatible strategies and can drive microbial diversification in ecological settings.

Abstract DNA methylation plays critical roles in gene regulation in bacteria, from regulating essential processes like the cell cycle to phenotypes of practical interest like pathogenicity and motility. Synthetic manipulation of global methylation levels has broad impacts on cellular physiology, changing expression patterns of hundreds of genes. However, whether or how environmental variation in natural settings similarly impacts DNA methylation patterns has been unclear. In this work, using the alphaproteobacteria Methylobacterium extorquens and Caulobacter crescentus as model systems, we discover the methylome is highly fluid in response to environmental variation, with different environments leading to distinct patterns of increased or decreased methylation levels along the chromosome. Despite a heterogeneous effect of different environments on methylation patterns, we find a general principle where the dependence of methylation states on position in the genome decreases in proportion to growth rate. A simple model that considers the methylation state through different phases of the cell cycle as a function of distance from an origin provides a framework to interpret the effects of different stressors upon the observed environmental responsiveness of the methylation patterns. Our work highlights how sequencing data alone can shed light on important aspects of microbial physiology. Significance Statement DNA methylation is known to profoundly impact gene regulation in prokaryotes, with both distinct methylation states at specific loci and global levels of DNA methylation modulating critical cellular phenotypes. Yet whether or how DNA methylation patterns depend on environmental variation remains unclear. Using Methylobacterium and Caulobacter as model systems, we combine experiments and theory to uncover general principles of how global patterns of DNA methylation are shaped by the environment. In particular, we discover a positive relationship between growth rate and the magnitude of the genomic position-dependent methylation level, highlighting how sequencing data can provide a culture-independent approach to estimating microbial traits like growth rate in natural settings. Our results resolve an open question, highlighting that DNA methylation patterns in bacteria can rapidly change in response to environmental shifts and revealing rules by which methylation patterns can help understand cellular phenotypes.

Lignin is a vast yet underutilized source of renewable energy. The microbial valorization of lignin is challenging due to the toxicity of its degradation intermediates, particularly formaldehyde. In this study, we engineered Methylobacterium extorquens PA1 to metabolize lignin-derived methoxylated aromatics, vanillate (VA) and protocatechuate (PCA), by introducing the van and pca gene clusters. Compared to Pseudomonas putida, M. extorquens PA1 exhibited better formaldehyde detoxification, enabling robust growth on VA without accumulation of formaldehyde. Genetic analyses confirmed that formaldehyde oxidation and stress response systems, rather than C1 assimilation, were important for VA metabolism. Additionally, VA and PCA were found to disrupt membrane potential, contributing to their inherent toxicity. Our findings establish M. extorquens PA1 as a promising chassis for lignin valorization and provide a framework for engineering formaldehyde-resistant microbial platforms.IMPORTANCEIn developing biotechnological solutions for a circular economy, it is critical to valorize all parts of renewable resources, such as lignocellulose from vegetative components of agricultural crops and from bioenergy feedstocks. After chemical breakdown, the aromatics arising from lignin present significant challenges for use due to their toxicity. Here, we address one component of this challenge-the methoxy groups that get released as formaldehyde-and show that existing biotechnological platform organisms with strong formaldehyde metabolism, such as Methylobacterium extorquens, can be transformed into highly capable utilizers of methoxylated aromatics.

Quantitative phase imaging (QPI) enables label-free microscopy with exceptional signal-to-background contrast and provides direct measurements of key biophysical parameters such as cell mass. Despite these advantages, QPI lacks a generalized, accurate, and computationally efficient segmentation method, limiting its use in high-throughput single-cell biology. Here, we introduce microbial Cell-Adaptive Segmentation (mCAS), a purpose-built framework for QPI. mCAS is an object-based, modular pipeline that leverages the robust phase-edge signal to achieve accurate segmentation without Fourier filtering, deconvolution, or deep learning. It runs efficiently on standard hardware, is readily implemented in common software platforms, and requires no curated training data. mCAS consistently outperformed both intensity-based and deep learning approaches, achieving over 98% accuracy with up to tenfold faster processing. By eliminating the segmentation bottleneck, mCAS enables scalable, high-fidelity analysis of QPI datasets and provides a broadly applicable foundation for label-free, high-throughput single-cell biology. We validate its generality across diverse bacterial species, morphologies, and intracellular complexities. Significance Statement Quantitative phase imaging (QPI) enables label-free, noninvasive measurement of cellular biophysics, yet its broader adoption has been limited by the absence of a generalized and computationally efficient segmentation strategy. We present microbial Cell-Adaptive Segmentation (mCAS), a framework purpose-built for QPI that achieves high accuracy without training, specialized hardware, or intensive preprocessing. mCAS generalizes across microbial species and morphologies, outperforming state-of-the-art deep learning approaches while processing thousands of single-cell images in minutes. By eliminating the segmentation bottleneck, mCAS makes QPI broadly accessible for high-throughput, label-free single-cell biology and expands the computational toolkit for quantitative imaging in microbiology. ### Competing Interest Statement The authors have declared no competing interest.

Bacteria leverage a secretion system to kill and scavenge nutrients from nearby competitors

Bacteria in the wild often encounter long periods of starvation. Evolutionary adaptation to cycles of nutrient deficiency has led to metabolic and physiological strategies that prevent cell death and promote rapid recovery from starvation. A cellular factor shaping outcomes during carbon starvation is polyhydroxybutyrate (PHB), a widespread carbon polymer that helps maintain membrane integrity and promotes regrowth upon reintroduction to nutrients. The response to starvation is often highly heterogeneous across isogenic populations and confounded by environmental variables that mask underlying physiological mechanisms. Prior work has shown that hyper- or hypo-osmolarity greatly increases cell death, but little is known about how PHB and osmolarity jointly influence both population viability and the emergence of heterogeneity in periods of nutrient limitation. To investigate this, we subjected populations of Methylobacterium extorquens to carbon starvation across a range of osmolarities and analyzed the phenotypes of individuals regrowing from the combined stress. By resolving the distributions of key traits such as lag time and growth rate, we found that populations partition into subpopulations that appear either significantly or minimally impaired by the stresses presented. Osmolarity affected total survival, and surprisingly, populations maintained viability in ultra-pure water. PHB metabolism was beneficial for population viability across all conditions, and unexpectedly reduced the variance of populations in lag time and growth rate. These findings show that PHB aids survival and regrowth from starvation, and suggest that the restructuring of phenotypes in long-term stationary phase depends on PHB even though total osmotic sensitivity does not. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, https://ror.org/021nxhr62, DBI-2320667 United States Department of Energy, https://ror.org/01bj3aw27, DE-SC0022318

Although the 16S (and 18S) rRNA gene has been an essential tool in classifying prokaryotes, using a single locus to revise bacteria taxonomy can introduce unwanted artifacts. There was a recent proposition to split the Methylobacterium genus, which contains diverse plant-associated strains and is important for agriculture and biotechnology, into two genera. Resting strongly on the phylogeny of 16S rRNA, 11 species of Methylobacterium were transferred to a newly proposed genus Methylorubrum. Numerous recent studies have independently questioned Methylorubrum as a valid genus, but the prior revision has left discrepancies among taxonomic databases. Here, we review phylogenomic and phenotypic evidence against Methylorubrum as a genus and call for its abandonment. Because Methylobacterium sensu lato forms a consistent and monophyletic genus, we argue for the restoration of the former and consensual Methylobacterium taxonomy. The large genomic, phenotypic, and ecological diversity within Methylobacterium however suggests complex evolutionary and adaptive processes and support the description of the most basal clade of Methylobacterium (group C) as a distinct genus in future work. Overall, this perspective demonstrates the danger of solely relying upon the 16S rRNA gene as a delimiter of genus level taxonomy and that further attempts must include more robust phenotypic and phylogenomic criteria.

Since its first demonstration over 100 years ago, scattering-based light-sheet microscopy has recently re-emerged as a key modality in label-free tissue imaging and cellular morphometry; however, scattering-based light-sheet imaging with subcellular resolution remains an unmet target. This is because related approaches inevitably superimpose speckle or granular intensity modulation on to the native subcellular features. Here, we addressed this challenge by deploying a time-averaged pseudo-thermalized light-sheet illumination. While this approach increased the lateral dimensions of the illumination sheet, we achieved subcellular resolving power after image deconvolution. We validated this approach by imaging cytosolic carbon depots in yeast and bacteria with increased specificity, no staining, and ultralow irradiance levels. Overall, we expect this scattering-based light-sheet microscopy approach will advance single, live cell imaging by conferring low-irradiance and label-free operation towards eradicating phototoxicity.

Combination antimicrobial therapy has been considered a promising strategy to combat the evolution of antimicrobial resistance. Francisella tularensis is the causative agent of tularemia and in addition to being found in the nature, is recognized as a threat agent that requires vigilance. We investigated the evolutionary outcome of adapting the Live Vaccine Strain (LVS) of F. tularensis subsp. holarctica to two non-interacting drugs, ciprofloxacin and doxycycline, individually, sequentially, and in combination. Despite their individual efficacies and independence of mechanisms, evolution to the combination arose on a shorter time scale than evolution to the two drugs sequentially. We conducted a longitudinal mutational analysis of the populations evolving to the drug combination, genetically reconstructed the identified evolutionary pathway, and carried out biochemical validation. We discovered that, after the appearance of an initial weak generalist mutation (FupA/B), each successive mutation alternated between adaptation to one drug or the other. In combination, these mutations allowed the population to more efficiently ascend the fitness peak through a series of evolutionary switch-backs. Clonal interference, weak pleiotropy, and positive epistasis also contributed to combinatorial evolution. This finding suggests that the use of this non-interacting drug pair against F. tularensis may render both drugs ineffective because of mutational switch-backs that accelerate evolution of dual resistance.

Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium is structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20 degrees C or 30 degrees C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100 km; between forests) and small (<1.2 km; within forests) geographical scales, among host tree species, and was dynamic over seasons. By measuring the growth of 79 isolates during different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together, our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This work paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus. IMPORTANCEMethylobacterium is a bacterial group tied to plants. Despite the ubiquity of methylobacteria and the importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and -independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial (within 1.2km) scales. Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains under laboratory conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasting growth strategies in response to environmental variations.

Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C1) compounds. The C1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. Furthermore, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium. We demonstrated growth on p-hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium, but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to Methylobacterium nodulans, and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation.

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum.

The phenomenon of phenotypic heterogeneity, where an isogenic population expresses varying phenotypes, has been uncovered for an expanding variety of organisms and traits. This heterogeneity in phenotype can be physiologically relevant, such as the ability for antibiotic persistence to allow populations to survive lethal conditions due to rare cells being in a slow or non-growing state. Recently, it was discovered that Methylorubrum extorquens , a facultative methylotroph, possesses a continuous spectrum of phenotypic states conferring tolerance to formaldehyde. Formaldehyde is a toxin produced by M. extorquens during growth on methanol. The phenotypic tolerance allowed rapid growth on levels of formaldehyde which were lethal to the majority of the population. Transcriptomics indicated this may be due to upregulation of proteins that attenuate oxidative stress and protein damage rather than increasing formaldehyde oxidation to prevent accumulation. These data suggested that heterogeneity to formaldehyde stress may be present even in non-methylotrophic organisms that do not routinely produce large quantities of formaldehyde, and thus widely distributed across bacteria. To investigate this, we tested Escherichia coli for heterogeneity to formaldehyde stress during growth on glucose. Like M. extorquens, E. coli populations have a wide, continuous range of formaldehyde tolerance thresholds and this tolerance was reversible. Several other features, however, were different from what was found for M. extorquens . Most E. coli growth occurred after formaldehyde levels had dropped, suggesting that persistence could be the cause. The dynamics of antibiotic persistence and formaldehyde tolerance were both tracked but found to not be correlated. On the other hand, the persister cell state can maintain formaldehyde tolerance. These data suggest that persistence can preserve phenotypic heterogeneities in other traits, further expanding its potential role in helping cells survive environmental stressors.

Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here, we present an overview of the module's learning goals, describe course resources (available for public use at http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

Understanding the evolution of novel physiological traits is highly relevant for expanding the characterization and manipulation of biological systems. Acquisition of new traits can be achieved through horizontal gene transfer (HGT). Here, we investigate drivers that promote or deter the maintenance of HGT-driven degeneracy, occurring when processes accomplish identical functions through nonidentical components. Subsequent evolution can optimize newly acquired functions; for example, beneficial alleles identified in an engineered Methylorubrum extorquens strain allowed it to utilize a "Foreign" formaldehyde oxidation pathway substituted for its Native pathway for methylotrophic growth. We examined the fitness consequences of interactions between these alleles when they were combined with the Native pathway or both (Dual) pathways. Unlike the Foreign pathway context where they evolved, these alleles were often neutral or deleterious when moved into these alternative genetic backgrounds. However, there were instances where combinations of multiple alleles resulted in higher fitness outcomes than individual allelic substitutions could provide. Importantly, the genetic context accompanying these allelic substitutions significantly altered the fitness landscape, shifting local fitness peaks and restricting the set of accessible evolutionary trajectories. These findings highlight how genetic context can negatively impact the probability of maintaining native and HGT-introduced functions together, making it difficult for degeneracy to evolve. However, in cases where the cost of maintaining degeneracy was mitigated by adding evolved alleles impacting the function of these pathways, we observed rare opportunities for pathway coevolution to occur. Together, our results highlight the importance of genetic context and resulting epistasis in retaining or losing HGT-acquired degenerate functions.

The recalcitrance of complex organic polymers such as lignocellulose is one of the major obstacles to sustainable energy production from plant biomass, and the generation of toxic intermediates can negatively impact the efficiency of microbial lignocellulose degradation. Here, we describe the development of a model microbial consortium for studying lignocellulose degradation, with the specific goal of mitigating the production of the toxin formaldehyde during the breakdown of methoxylated aromatic compounds. Included are Pseudomonas putida, a lignin degrader; Cellulomonas fimi, a cellulose degrader; and sometimes Yarrowia lipolytica, an oleaginous yeast. Unique to our system is the inclusion of Methylorubrum extorquens, a methylotroph capable of using formaldehyde for growth. We developed a defined minimal “Model Lignocellulose” growth medium for reproducible coculture experiments. We demonstrated that the formaldehyde produced by P. putida growing on vanillic acid can exceed the minimum inhibitory concentration for C. fimi, and, furthermore, that the presence of M. extorquens lowers those concentrations. We also uncovered unexpected ecological dynamics, including resource competition, and interspecies differences in growth requirements and toxin sensitivities. Finally, we introduced the possibility for a mutualistic interaction between C. fimi and M. extorquens through metabolite exchange. This study lays the foundation to enable future work incorporating metabolomic analysis and modeling, genetic engineering, and laboratory evolution, on a model system that is appropriate both for fundamental eco-evolutionary studies and for the optimization of efficiency and yield in microbially-mediated biomass transformation.